P62/SQSTM1 binds with claudin-2 to target for selective autophagy in stressed intestinal epithelium

Impaired autophagy promotes Inflammatory Bowel Disease (IBD). Claudin-2 is upregulated in IBD however its role in the pathobiology remains uncertain due to its complex regulation, including by autophagy. Irrespective, claudin-2 expression protects mice from DSS colitis. This study was undertaken to examine if an interplay between autophagy and claudin-2 protects from colitis and associated epithelial injury. Crypt culture and intestinal epithelial cells (IECs) are subjected to stress, including starvation or DSS, the chemical that induces colitis in-vivo. Autophagy flux, cell survival, co-immunoprecipitation, proximity ligation assay, and gene mutational studies are performed. These studies reveal that under colitis/stress conditions, claudin-2 undergoes polyubiquitination and P62/SQSTM1-assisted degradation through autophagy. Inhibiting autophagy-mediated claudin-2 degradation promotes cell death and thus suggest that claudin-2 degradation promotes autophagy flux to promote cell survival. Overall, these data inform for the previously undescribed role for claudin-2 in facilitating IECs survival under stress conditions, which can be harnessed for therapeutic advantages.

In this paper Ahmad et al. showed that the treatment of intestinal epithelial cells with colitoge or upon starvation decreases the plasma membrane protein Claudin-2, and it relies on autophagy and selective autophagy receptor p62. They claimed that Lys218 of Claudin-2 is ubiquitinated at Lys63linkage upon the treatment of colitoge, which is indispensable for downregulation of Claudin-2. The defect of this pathway caused activation of caspase both in vitro and in vivo. Although some of the results are interesting, there are several scattered points where the interpretation of the experimental results is not well founded. Furthermore, the experiments are insufficient to prove the authors' claims.
1. Caludin-2 is a plasma membrane protein and to undergo degradation by ATG16L or p62dependent autophagy (macroautophagy), Caludin-2 should be pulled from the plasma membrane or vesicles containing Caludin-2 should be formed from the plasma membrane. There is no experimental approach to this point, and it is not mentioned even in the Discussion section. 2. To prove p62-dependent degradation of Claudin-2, knockdown experiments targeting p62 and immunoprecipitation assays with a p62 mutant deleting the UBA domain are required. 3. The results with PR-619 were incomprehensible. The PR-619 treatment would cause an increase in ubiquitinated proteins since it is an inhibitor of broad range of deubiquitinating enzyme inhibitor. Nevertheless, the authors appear to be using PR-619 as an inhibitor of ubiquitination. Such experiments would normally be suitable for the use of a cell membrane-permeable ubiquitin activating enzyme (E1) inhibitor (e.g., UBEI-41). If the authors use deubiquitination or ubiquitination inhibitors, immunoblots with ubiquitin antibody should be presented to ensure their effectiveness. 4. The data shown in Fig. 4E-F merely show that there is Claudin-2ubiquitinylated at the K63linkage, but do not rule out ubiquitination at other linkages such as K48. 5. Fig. 5C-E is also difficult to interpret: if immunoprecipitation is performed with a K63-linkagespecific ubiquitin antibody and then the immunoprecipitates are analyzed by immunoblot with Claudine-2 antibody, the Claudine-2 detected should be ubiquitinated one. However, the detected one appears to be free Claudin-2, which is not ubiquitinated. 6. Fig. 2G and H does not mean autophagy-flux by mere blotting of LC3; autophagic flux should be verified using inhibitors such as Baf. A1. 7. Fig. 2A, why does actinomycin D treatment cancel the effect of DSS (Claudin-2 decrease)?
Reviewer #2 (Remarks to the Author): This manuscript mainly studied the role of Claudin-2 in the DSS-induced autophagy, and found that DSS/starvation induces the degradation of Claudin-2 by P62/LC3-dependent manner. Authors also identified the specific ubiquitination site of Claudin-2. Overall, it presents a novel target of P62-dependent selective autophagy, and demonstrates that the degradation of Claudin-2 is associated with cell death induced by DSS/starvation. Concerns: 1) The association between degradation of Claudin-2 and cell death is missing. What is the mechanism of cell death induced by degradation of Claudin-2? Authors need to further explore the mechanism of Claudin-2 degradation and cell death.
2) The quality of data is poor. For example, in Fig 4F, there isn't obvious increasment of ubiquitination of Claudin-2 after DSS treatment; In Fig 5D, there isn't ubiquitination of Claudin-2, and arrow indicated band might be none-specific band; In Fig 5E, there isn't any difference in ubiquitination of Claudin-2 between wild-type and mutant Claudin-2. Authors need to present data with higher quality, and some of the present data aren't convincing.
3) What's the role of Claudin-2 in DSS-induced cell death? promotion or prevention? As in Fig 6G, authors showed that over-expression of Claudin-2 prevented cell death. While in Fig 6H, K218 mutant of Claudin-2 (stable mutant) promote cell death. So the results are confusing. 4) Since authors have Claudin-2 transgenic mice and KO mice, it would be important to examine the role of Claudin-2 in cell death by in vivo model, for example, DSS-induced colitis model.

Reviewer #3 (Remarks to the Author):
The authors investigated the interaction between Claudin2 and P62/SQSTM1 and found that DSS can affect the ubiquitination degradation of Claudin2 through autophagy, which has a certain research value, but there are still some minor problems： 1.It is still controversial to directly treat cells with DSS to simulate chemically induced colitis model, and the mechanism of DSS-induced colitis animal model has not been fully clarified at present. It is suggested that the authors alternatively choose IECs treated with complex inflammatory factors (TNF-a and IFN-γ, etc.) for further verification. 2.Several WB bands of β-Actin show multiple bands (as shown in Fig.1A and 1B). Please provide the original pictures and explanations. 3.In Fig.3C and 3D, it is suggested to add the Input group in co-IP. 4.As suggested by the authors, the elevated level of claudin2 in IBD patients in previous studies is inconsistent with the DSS-induced mice and IEC in this article, and it is recommended to add human IBD samples to verify the levels of claudin-2, autophagy, and related molecules.
We are submitting the revised version of our original article . Due to the extensive revisions and COVID-related supply chain issues and personal sickness, it took longer than anticipated in submitting this revised article. We would like to sincerely thank the reviewers for their time and effort in reviewing our manuscript and providing highly constructive comments and suggestions. We have modified the manuscript to the best of our capacity to address the reviewer's comments, which we believe has strengthened the manuscript. We hope that the revised article is considered suitable for publication in Communication Biology.
Below, please find the reviewer's comments summarized in black font, followed by our responses in blue font. Corresponding changes made to the manuscript text are shown in red font.

Reviewer #1 (Remarks to the Author):
Query#1. Caludin-2 is a plasma membrane protein and to undergo degradation by ATG16L or p62dependent autophagy (macroautophagy), Caludin-2 should be pulled from the plasma membrane or vesicles containing Caludin-2 should be formed from the plasma membrane. There is no experimental approach to this point, and it is not mentioned even in the Discussion section.
Response: Reviewer has raised an excellent point. Published studies have demonstrated that claudin-2 protein from the membrane can be trafficked by Rab14, clathrin, and caveolin-1, which can be recycled back to the membrane or degraded in the lysosomes (PMID:26163137; 21660968; 22396724; 25694446; 30899070). Moreover, recently Ganapathy et al. showed that AP2M1 mediates autophagyinduced claudin-2 degradation through endocytosis (PMID: 34964704). We have discussed these possibilities in the discussion part in the revised manuscript.
Query#2. To prove p62-dependent degradation of Claudin-2, knockdown experiments targeting p62 and immunoprecipitation assays with a p62 mutant deleting the UBA domain are required.

Response:
As per reviewer's suggestion, we now provide new data that genetic silencing of P62 expression (siRNA mediated) prevents both, DSS-and starvation-mediated downregulation of claudin-2 expression. These data are included in the revised manuscript ( Figure 3G).
Query#3. The results with PR-619 were incomprehensible. The PR-619 treatment would cause an increase in ubiquitinated proteins since it is an inhibitor of broad range of deubiquitinating enzyme inhibitor. Nevertheless, the authors appear to be using PR-619 as an inhibitor of ubiquitination. Such experiments would normally be suitable for the use of a cell membrane-permeable ubiquitin activating enzyme (E1) inhibitor (e.g., UBEI-41). If the authors use deubiquitination or ubiquitination inhibitors, immunoblots with ubiquitin antibody should be presented to ensure their effectiveness.

Response:
We appreciate the reviewer's constructive criticism and as suggested by the reviewer, we repeated our studies using PYR41, a cell-permeable ubiquitin-activating enzyme inhibitor. We also performed analysis of K63-linked ubiquitination under the same setting. In the revised manuscript, we have provided the new data as Figure 4A and 4B.
Query#4. The data shown in Fig. 4E-F merely show that there is Claudin-2 ubiquitinylated at the K63linkage, but do not rule out ubiquitination at other linkages such as K48.

Response:
As per reviewer's suggestion, we performed parallel co-immunoprecipitation using lysates from control and DSS-treated Caco-2 cells and antibodies specific for K48 and K63 linked ubiquitin. The outcome demonstrated robust association of claudin-2 with K63-linked, however also a minor association of claudin-2 with K-48 linked ubiquitin was also observed. We have added these data as Figure 4E and Supplementary Figure 3 in the revised manuscript.
Query#5. Fig. 5C-E is also difficult to interpret: if immunoprecipitation is performed with a K63-linkagespecific ubiquitin antibody and then the immunoprecipitates are analyzed by immunoblot with Claudine-2 antibody, the Claudine-2 detected should be ubiquitinated one. However, the detected one appears to be free Claudin-2, which is not ubiquitinated.

Response:
We repeated these studies using the same claudin-2 mutant constructs. Caco-2 cells where these constructs were overexpressed were then subjected to the DSS-treatment. The overexpressed claudin-2 protein was immunoprecipitated using anti-HA-Tag antibody followed by immunoblotting with anti-claudin-2 and -K63 ubiquitin antibodies. The mutant claudin2K218A HA exhibited markedly reduced K63-linked ubiquitination of claudin-2. This data is provided in revised manuscript as Figure 5D.
Query#6. Fig. 2G and H does not mean autophagy-flux by mere blotting of LC3; autophagic flux should be verified using inhibitors such as Baf. A1.

Response:
As per reviewer's suggestion, we analyzed autophagy flux using Baf A1 in Caco-2 cells subjected to the DSS treatment. The outcome has been added to the revised manuscript as Figure 2I) Query#7. Fig. 2A, why does actinomycin D treatment cancel the effect of DSS (Claudin-2 decrease)?
Response: We appreciate this insightful comment. In our detailed analysis, we found that the DSStreatment also affects claudin-2 mRNA expression. However, this effect is significantly lower compared to the effects of stress/colitogens on claudin-2 protein expression (Supplementary Figure 2A). Thus, the slight rescue of the DSS-mediated claudin-2 downregulation by Actinomycin-D treatment was in the line of our additional data.

Reviewer #2:
Query#1. The association between degradation of Claudin-2 and cell death is missing. What is the mechanism of cell death induced by degradation of Claudin-2? Authors need to further explore the mechanism of Claudin-2 degradation and cell death.

Response:
Increased autophagy flux supports cell survival under stress conditions. Our data suggest that claudin-2 serves as a substrate for selective autophagy. Our data that cells overexpressing claudin-2 mutant unable to undergo autophagic degradation also undergo apoptosis supports that autophagicdegradation of claudin-2 promotes cells survival. Mechanistic details of whether claudin-2 degradation also promotes other cell survival mechanisms are plausible and part of our ongoing studies.
2) The quality of data is poor. For example, in Fig 4F, there isn't obvious increasment of ubiquitination of Claudin-2 after DSS treatment; In Fig 5D, there isn't ubiquitination of Claudin-2, and arrow indicated band might be none-specific band; In Fig 5E, there isn't any difference in ubiquitination of Claudin-2 between wild-type and mutant Claudin-2. Authors need to present data with higher quality, and some of the present data aren't convincing.
promote the autophagic flux and associated cell survival. Thus, when claudin-2 is mutated to not participate in autophagy, cells will undergo apoptosis as overexposed mutant claudin-2 is not available for the autophagic degradation and thus promoting autophagy flux. 4) Since authors have Claudin-2 transgenic mice and KO mice, it would be important to examine the role of Claudin-2 in cell death by in vivo model, for example, DSS-induced colitis model.

Response:
We have previously reported that claudin-2 transgenic mice are protected against DSScolitis and associated epithelial cell death (PMID#24670427). Another group has reported that claudin-2 KO mice develop severe colitis and epithelial cell death when subjected to DSS-colitis (PMID# 23306855).

Reviewer #3:
Query#1. It is still controversial to directly treat cells with DSS to simulate chemically induced colitis model, and the mechanism of DSS-induced colitis animal model has not been fully clarified at present. It is suggested that the authors alternatively choose IECs treated with complex inflammatory factors (TNF-a and IFN-γ, etc.) for further verification.
Response: Several recent studies on intestinal inflammation have reported DSS-treatment as the tool to induce intestinal epithelial cell injury (some of them-PMID#: 35495925; 30476915; 17089061, and 29396428). By directly exposing epithelial cells to DSS in-vitro conditions, we create a similar milieu as in the in-vivo model. Moreover, we have also treated cells with TNBS, yet another colitogen. Treating IECs with complex inflammatory factors such as TNF-α and IFN-γ would not serve our purpose as it would involve inflammatory signaling. Apart, throughout the study, we have used starvation as complementary model of stress induced epithelial injury.
Query#2. Several WB bands of β-Actin show multiple bands (as shown in Fig.1A and 1B). Please provide the original pictures and explanations.

Response:
We regret the double band of β-Actin pointed out by the reviewer. However, sometimes multiple bands do appear during immunoblot development using some β-Actin antibodies. We have provided improved blots to replace these blots ( Figure 1A and 1B).
Query#3. In Fig.3C and 3D, it is suggested to add the Input group in co-IP.

Response:
In these figures, the inherent goal is to analyze physical interaction by the immunedepletion method. For this, we used increasing claudin-2 or P62 antibody concentration to deplete the interacting partner in the lysate. We ran two different gels; one gel confirmed that immune depletion worked, and the second showed a similar decrease in the amount of both P62 and claudin-2 when we depleted either one. However, as per the reviewer's suggestions, we repeated the experiment and replaced Figures 3C and 3D in the revised manuscript.
Query#4. As suggested by the authors, the elevated level of claudin2 in IBD patients in previous studies is inconsistent with the DSS-induced mice and IEC in this article, and it is recommended to add human IBD samples to verify the levels of claudin-2, autophagy, and related molecules.
Response: Autophagy is dysregulated in IBD patients (PMID#17921695; 17435756), which may explain why claudin-2 is upregulated in IBD. We performed co-immunofluorescence using anti-claudin-2, LC3, and P62 antibodies in IBD patient samples. The outcome showed claudin-2 immunolocalization that is dysregulated from its membrane-tethered localization which co-localized with LC3 and P62. These results have been included in the revised manuscript ( Figure 7A and 7B).